The molecular mechanism through which cholesterol and oxygenated sterols regulate the rate of cholesterol synthesis by controlling the level of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase will be determined. We have adapted established liver cell lines of rat and human (ATCC # CCL 13) origin to growth in monolayer and suspension culture in serum and cholesterol-free medium and have isolated clonal lines of mutant cells selected by resistance to oxygenated sterols, or to the competitive inhibitor of HMG-CoA reductase, ML-236B (compactin). We shall isolate a third class of variant defective in regulation by cholesterol. We shall determine whether mutants which overproduce HMG-CoA reductase are gene amplification mutants or are regulatory mutants by quantitation of HMG-CoA reductase mRNA levels and of the number of HMG-CoA reductase NDA from normal and mutant cells. The site(s) and mechanism(s) of action of cholesterol and oxygenated sterols will be identified with a view to determining whether they exert their effects on HMG-CoA reductase levels through a common regulatory mechanism. Monclonal antibody against HMG-CoA reductase will be employed in immunochemical studies of the rates of HMG-CoA reductase synthesis and degradation in normal and mutant cells exposed to cholesterol or oxygenated sterols. Immunoprecipitations will employ a very low background preformed indirect immunoprecipitation method we have recently developed. Levels of HMG-CoA reductase mRNA will be determined--initially by in vitro translation and subsequently by hybridization using cloned HMG-CoA reductase cRNA fragments. Hybridization of pulse labeled RNA to cloned reductase cDNA immobilized on nitrocellulose filters will be employed to determine whether transcription, nuclear processing and exit, or cytoplasmic mRNA degradation are implicated in control of reductase mRNA levels. The location, number, and size of HMG-CoA reductase synthesizing polysomes and the possible existence of a hydrophobic signal peptide or other precursor sequence will be examined by mRNA translation.